1. Field of the Invention
Cryopreservation methods exist which adequately preserve germplasm and embryos from most livestock. Pig embryos are an exception, however, since whole early stage pig embryos up to the morula stage do not develop after conventional cryopreservation. There has thus been a strong incentive to develop a method which allows effective preservation of embryos from these animals at all stages of embryological development. This invention relates to a method for cryopreservation of swine embryos at the morula/blastocyst stage utilizing vitrification or freezing. The process allows effective, long-term storage of embryos for subsequent transfer to recipient females.
2. Description of the Relevant Art
Boar sperm cryopreservation methods have been available since 1975 and utilized sparingly in commercial production. There has been little success in preserving pig oocytes and embryos by conventional freezing methods, however. The cryopreservation of embryos in addition to sperm thus represents a potential increase in the efficiency of transmitting improved genetic potential.
Pig embryos suffer from severe sensitivity to hypothermia (Wilmut, I. 1972. J. Reprod. Fertil. vol. 31, pp. 513-514; Polge et al. 1974. Cryobiology. vol. 11, p. 560; Polge and Willadsen. 1978. Cryobiology. vol. 15, pp. 370-373; Pollard and Leibo. 1994. Theriogenology. vol. 41, pp. 101-106; all herein incorporated by reference) which limits their ability to withstand cryopreservation. A high lipid content is believed to be the critical factor in hypothermic sensitivity and cryosurvival.
While numerous studies have reported on conventional freezing methods, vitrification has more recently shown potential for avoiding the problems associated with cooling sensitivity and ice crystallization (Dobrinsky, J. R. 1998. J. Reprod. Fert. suppl. 51, pp. 301-312; Dobrinsky et al. 2000. Biology of Reproduction. vol. 62, pp. 564-570; both herein incorporated by reference). Cattle embryos, for example, have been successfully cryopreserved by vitrification (Massip et al. 1986. Cryo-Letters. vol. 7, pp. 270-273; Dobrinsky et al. 1991. Theriogenology. vol. 35, p. 194; both herein incorporated by reference), and van Wagtendonk-de Leeuw et al. (1997. Theriogenology. vol. 48, pp. 1071-1084, herein incorporated by reference) showed that bovine embryo vitrification could be successfully applied under field conditions without a reduction in pregnancy rate. Dobrinsky and Johnson (1994. Theriogenology. vol. 42, pp. 25-35, herein incorporated by reference) first demonstrated the efficacy of vitrification of swine embryos where survival and subsequent development in vitro could be established (up to 40% survival). Embryo survival, however, was limited to expanded and early hatched blastocyst stage embryos. It has been shown that hatched blastocyst pig embryos can survive vitrification and, after transfer, can go on to develop in vivo to term (Dobrinsky et al. 1998. Theriogenology. vol. 49, p. 166; Kobayashi et al. 1998. Cryobiology. vol. 36, pp. 20-31; both herein incorporated by reference). The numbers of recipients in these studies were low, however, and they have not been repeated. Most recently, Dobrinsky et al. (2000, supra) have shown that vitrified hatched blastocyst stage embryos are capable of surviving preservation with the production of live offspring after vitrification, warming and embryo transfer.
Although success with the cryopreservation of hatched blastocysts has been achieved and reported in the literature, embryos preserved at the hatched blastocyst stage of development cannot be utilized globally, fresh or cryopreserved under conventional commercial circumstances. Hatched blastocysts have lost the zona pellucida, or outer layer, by that stage of development, and it is a requirement for the international movement of embryos that they be zona pellucida-intact (Manual of the International Embryo Transfer Society, 3rd ed. (1998). The zona pellucida surrounds the embryo from the oocyte to the late blastocyst (hatched blastocyst) stage. During fertilization, it acts as a barrier to polyspermic insemination, allowing only one sperm to penetrate and thus fertilize the oocyte. The matrix also acts as an artifactual barrier to pathogens that may be present in the uterus of the mother. These pathogens may infect the embryo after hatching from the zona pellucida has occurred, thus transporting pathogens from the mother to the embryo which could in turn possibly infect a recipient animal. The search has therefore continued for a method for the effective cryopreservation of swine embryos having intact zona pellucidae.
We have discovered a novel process for the cryopreservation of swine embryos which is effective at the morula/blastocyst stage of development, at which stage the zona pellucida is intact. The method requires a centrifugation step prior to cryopreservation which allows lipids distributed throughout the embryo to become sufficiently localized that damage during cryopreservation to the embryo caused by the presence of intracellular lipids is thereby avoided. Further, after vitrification and warming or freezing and thawing, as appropriate following cryopreservation, the zona pellucida is removed from the embryo before transfer into a recipient female. The embryos then continue normal development in vivo.
In accordance with this discovery, it is an object of the invention to provide a method for cryopreserving swine embryos having intact zona pellucidae. The cryopreserved embryos may be further processed for embryo transfer to a recipient female.
It is also an object of the invention to provide a cryopreserved swine embryo having an intact zona pellucida which is effective for embryo transfer.
Other objects and advantages of the invention will become readily apparent from the following description.